Synaptic transmission involves the transformation of electrical into chemical signals by the regulated release of neurotransmitter from synaptic vesicles. High frequency synaptic transmission depends on the recycling of neurotransmitter back into the nerve terminal after release, where it is re-packaged into synaptic vesicles by vesicular neurotransmitter transporter proteins located in the vesicle membrane. Repetitive neurotransmitter release also requires mechanisms to recycle synaptic vesicle membrane and proteins locally at the nerve terminal. Variation in the kinetics of synaptic vesicle recycling may shape the amount and pattern of neurotransmitter output, and hence contribute to information processing and synaptic plasticity. The distribution of vesicles in functional pools, defined by their location and fusion probability, can also influence neurotransmitter release in response to repetitive firing. Multiple pathways have been proposed for the recycling of synaptic vesicle components after exocytosis, but the relationship of these pathways to the different synaptic vesicle pools has remained unclear. Synaptic vesicle proteins have been assumed to undergo recycling as a unit. However, emerging data indicates that differences in the association with distinct endocytic adaptor proteins may influence the trafficking of individual synaptic vesicle proteins, affecting the composition of synaptic vesicles and hence their functional characteristics. The long-term goal of the proposed research is to understand how membrane trafficking of individual vesicular proteins influences the protein composition of synaptic vesicles, the maintenance of synaptic vesicle pools, and the release of transmitter by specific circuits. The strategy of this proposal is to study the trafficking of synaptic vesicle proteins by a combination of biochemistry, live cell imaging, electrophysiology, and electron microscopy. Fusions of vesicular neurotransmitter transporters with pH-sensitive fluorescent proteins provide optical probes to study the effect of protein interactions and regulatory mechanisms on the recycling pathways of vesicular transporter proteins. The specific aims of this proposal are designed to study the regulation of trafficking of vesicular glutamate transporters VGLUT1 and 2 by 1) characterizing differences in the trafficking of two closely related isoforms, 2) identifying the protein determinants and 3) biochemical and cellular mechanisms underlying VGLUT recycling. Regulation of neurotransmitter release may be an important approach to therapeutic intervention and the molecular machinery new targets for the development of better treatments for neuropsychiatric disorders such as epilepsy, schizophrenia, and anxiety disorders.